High Developmental Potential Research Articles

The kinetics of nucleolar precursor bodies clustering at the pronuclei interface: Positive correlations with the morphokinetic characteristics of cleaving embryos and euploidy in preimplantation genetic testing programs

Objective: This study investigated potential relationships between the kinetics of nucleolar precursor bodies (NPBs) in the pronucleus and developmental morphokinetics and euploidy in human preimplantation genetic testing for aneuploidy (PGT-A) cycles.Methods: The morphokinetic analysis of 200 blastocysts obtained from 53 PGT-A cycles was performed retrospectively in a time-lapse incubator. At the time of pronuclear breakdown (PNBD), we categorized the blastocysts into two groups based on the kinetic degree of clustering NPBs at the interface of the two pronuclei: clustered NPBs (CL) and non-clustered NPBs (NCL). We then compared morphokinetic parameters, abnormal behavioral events, and the rate of aneuploidy between the two groups.Results: Pronuclear fading and the first cleavage occurred earlier in the NCL group than in the CL group. However, the initiation of blastocyst formation and blastocyst expansion was delayed in the NCL group relative to the CL group. No differences were found in the rate of abnormal cleavage events, such as multinucleation at the 2-cell stage, direct cleavage from one to three cells, and from two to five cells between the CL and NCL groups. However, the fragmentation rate at the 8-cell stage was higher in the NCL group than in the CL group (10.3% vs. 1.9%, p<0.05). Additionally, the euploid rate in the CL group was significantly higher than in the NCL group (37.9% vs. 12.4%, p<0.05).Conclusion: These results demonstrate the effectiveness of combining NPB clustering at PNBD with morphokinetics as a parameter for selecting embryos with higher developmental potential in in vitro fertilization.

Substrates mimicking the blastocyst geometry revert pluripotent stem cell to naivety.

Naive pluripotent stem cells have the highest developmental potential but their in vivo existence in the blastocyst is transient. Here we report a blastocyst motif substrate for the in vitro reversion of mouse and human pluripotent stem cells to a naive state. The substrate features randomly varied microstructures, which we call motifs, mimicking the geometry of the blastocyst. Motifs representing mouse-blastocyst-scaled curvature ranging between 15 and 62 mm-1 were the most efficient in promoting reversion to naivety, as determined by time-resolved correlative analysis. In these substrates, apical constriction enhances E-cadherin/RAC1 signalling and activates the mechanosensitive nuclear transducer YAP, promoting the histone modification of pluripotency genes. This results in enhanced levels of pluripotency transcription factor NANOG, which persist even after cells are removed from the substrate. Pluripotent stem cells cultured in blastocyst motif substrates display a higher development potential in generating embryoid bodies and teratomas. These findings shed light on naivety-promoting substrate design and their large-scale implementation.

O-171 Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo

Abstract Assessing the morphological appearance of an embryo as it progresses from zygote to blastocyst is the primary method used by embryologists to evaluate embryo viability and developmental potential. Standard embryo grading techniques, using time-lapse microscopy, analyse morphological characteristics such as size, shape, number, and appearance of blastomeres. The aim of this study was to determine how individual cells of the embryo function on a subcellular level to build a healthy embryo. RNA localisation plays indispensable roles in establishing asymmetries and coordinating cell fate decisions during early embryogenesis across various non-mammalian species. To direct the spatiotemporal distribution of RNA within the cells of an embryo, the microtubule cytoskeleton provides highly sophisticated trafficking pathways. Yet, it remains unknown whether subcellular heterogeneities exist during mammalian preimplantation development and how they contribute to cell fate determination. Using advanced live imaging, we visualised global RNA transcripts at high spatiotemporal resolution from fertilisation to the blastocyst stage in living preimplantation mouse embryos. We discovered apicobasal RNA asymmetries specific to outer cells of the 16-cell stage mouse embryo, coinciding with cell fate decisions and the emergence of the pluripotent inner cell mass. Highly clustered RNAs accumulated proximal to the basal membrane, while more dispersed RNA foci were identified apically as the embryo reaches the late 16-cell stage. The targeted distribution of membrane-less RNA molecules was facilitated by the microtubule cytoskeleton, associated with lysosomes which serve as RNA transport vehicles. We discovered that apically located RNA foci were more dynamic and accompanied an enrichment of translation components. Furthermore, we uncovered that the established RNA asymmetries enhanced translation capacity in apical regions compared to basal regions. These spatiotemporal RNA heterogeneities were unevenly inherited by outer and inner daughter cells during subsequent cell divisions. Notably, embryos that were unable to establish RNA asymmetries failed to allocate cells to the trophectoderm and inner cell mass. Our study provides insights into a subcellular mechanism driving asymmetric RNA localisation and compartmentalised translational regulation in outer cells of the 16-cell stage embryo, which may contribute to cell fate decisions during mammalian preimplantation embryogenesis. We envision that when paired with classical morphological assessment criteria of the embryo, these findings may assist with selecting the embryo with the highest developmental potential.

In vitro matured oocytes have a higher developmental potential than in vivo matured oocytes after hormonal ovarian stimulation in Callithrix jacchus

BackgroundThe common marmoset, Callithrix jacchus, is an invaluable model in biomedical research. Its use includes genetic engineering applications, which require manipulations of oocytes and production of embryos in vitro. To maximize the recovery of oocytes suitable for embryo production and to fulfil the requirements of the 3R principles to the highest degree possible, optimization of ovarian stimulation protocols is crucial. Here, we compared the efficacy of two hormonal ovarian stimulation approaches: 1) stimulation of follicular growth with hFSH followed by triggering of oocyte maturation with hCG (FSH + hCG) and 2) stimulation with hFSH only (FSH-priming).MethodsIn total, 14 female marmosets were used as oocyte donors in this study. Each animal underwent up to four surgical interventions, with the first three performed as ovum pick-up (OPU) procedures and the last one being an ovariohysterectomy (OvH). In total, 20 experiments were carried out with FSH + hCG stimulation and 18 with FSH-priming. Efficacy of each stimulation protocol was assessed through in vitro maturation (IVM), in vitro fertilization (IVF) and embryo production rates.ResultsEach study group consisted of two subgroups: the in vivo matured oocytes and the oocytes that underwent IVM. Surprisingly, in the absence of hCG triggering some of the oocytes recovered were at the MII stage, moreover, their number was not significantly lower compared to FSH + hCG stimulation (2.8 vs. 3.9, respectively (ns)). While the IVM and IVF rates did not differ between the two stimulation groups, the IVF rates of in vivo matured oocytes were significantly lower compared to in vitro matured ones in both FSH-priming and FSH + hCG groups. In total, 1.7 eight-cell embryos/experiment (OPU) and 2.1 eight-cell embryos/experiment (OvH) were obtained after FSH + hCG stimulation vs. 1.8 eight-cell embryos/experiment (OPU) and 5.0 eight-cell embryos/experiment (OvH) following FSH-priming. These numbers include embryos obtained from both in vivo and in vitro matured oocytes.ConclusionA significantly lower developmental competence of the in vivo matured oocytes renders triggering of the in vivo maturation with hCG as a part of the currently used FSH-stimulation protocol unnecessary. In actual numbers, between 1 and 7 blastocysts were obtained following each FSH-priming. In the absence of further studies, FSH-priming appears superior to FSH + hCG stimulation in the common marmoset under current experimental settings.

Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, invivo (high developmental potential (IV)), invitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to invivo versus invitro and invivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between invitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between invivo and cloned conceptuses, while only 6 miRNAs were different between invivo and invitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium invivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

How great thou ART: biomechanical properties of oocytes and embryos as indicators of quality in assisted reproductive technologies.

Assisted Reproductive Technologies (ART) have revolutionized infertility treatment and animal breeding, but their success largely depends on selecting high-quality oocytes for fertilization and embryos for transfer. During preimplantation development, embryos undergo complex morphogenetic processes, such as compaction and cavitation, driven by cellular forces dependent on cytoskeletal dynamics and cell-cell interactions. These processes are pivotal in dictating an embryo's capacity to implant and progress to full-term development. Hence, a comprehensive grasp of the biomechanical attributes characterizing healthy oocytes and embryos is essential for selecting those with higher developmental potential. Various noninvasive techniques have emerged as valuable tools for assessing biomechanical properties without disturbing the oocyte or embryo physiological state, including morphokinetics, analysis of cytoplasmic movement velocity, or quantification of cortical tension and elasticity using microaspiration. By shedding light on the cytoskeletal processes involved in chromosome segregation, cytokinesis, cellular trafficking, and cell adhesion, underlying oogenesis, and embryonic development, this review explores the significance of embryo biomechanics in ART and its potential implications for improving clinical IVF outcomes, offering valuable insights and research directions to enhance oocyte and embryo selection procedures.

Ethnobotanical study of traditional forage plants in the Gansu–Ningxia–Inner Mongolia junction zone: conservation and sustainable utilization for animal husbandry

IntroductionThis study aims to safeguard the ethnobotanical knowledge pertaining to traditional forage plants within the ethnically diverse Gansu–Ningxia–Inner Mongolia junction zone. It seeks to establish a foundation for the sustainable utilization of these traditional resources for animal husbandry.MethodsA combination of literature research, village interviews, participatory observation, and ethnobotanical quantitative evaluation methods was employed to investigate and study the traditional knowledge of wild forage plants used by local residents in the study area.ResultsLocal residents provided information on 73 forage plants, which were identified as 116 distinct wild forage plant species. These plants belong to 22 families and play an active role in the lives of the local inhabitants. Notably, the families Poaceae, Fabaceae, and Asteraceae are prominent, comprising the most abundant and widely utilized wild forage plants. Bing Cao (collectively referring to plants of the Agropyron, Leymus, and Psammochloa), Suo Cao (collectively referring to plants of the genus Stipa), and Ku Cai (encompassing Lactuca tatarica (L.) C.A.Mey. and Ixeris polycephala Cass.) emerge as the most representative and vital wild forage plants for animal husbandry. Additionally, plants within the Astragalus (referred to collectively as NiaoZi by local residents) in the Fabaceae family, as well as plants from the Amaranthaceae family, exhibit notable significance.ConclusionAnimal husbandry assumes a pivotal role in the local agricultural economy, and the 116 wild forage plants investigated hold substantial importance in its development. Among these, 59 and 103 plant resources display high developmental potential, making them prospective candidates for high-quality cultivated forage grasses. Additionally, extensive grazing practices have resulted in significant ecological degradation within this already fragile ecosystem. The cultivation of forage grasses and the practice of pen-based animal husbandry may emerge as crucial strategies for sustainable development in this area.

Human oocytes image classification method based on deep neural networks

BackgroundThe effectiveness of in vitro fertilization depends on the assessment and selection of oocytes and embryos with the highest developmental potential. One of the tasks in the ICSI (intracytoplasmic sperm injection) procedure is the classification of oocytes according to the stages of their meiotic maturity. Oocytes classification traditionally is done manually during their observation under the light microscope. The paper is part of the bigger task, the development of the system for optimal oocyte and embryos selection. In the hereby work, we present the method for the automatic classification of oocytes based on their images, that employs DNN algorithms.ResultsFor the purpose of oocyte class determination, two structures based on deep neural networks were applied. DeepLabV3Plus was responsible for the analysis of oocyte images in order to extract specific regions of oocyte images. Then extracted components were transferred to the network, inspired by the SqueezeNet architecture, for the purpose of oocyte type classification. The structure of this network was refined by a genetic algorithm in order to improve generalization abilities as well as reduce the network’s FLOPs thus minimizing inference time. As a result, overline{Acc} at the level of 0.964 was obtained at the level of the validation set and 0.957 at the level of the test set. Generated neural networks as well as code that allows running the processing pipe were made publicly available.ConclusionsIn this paper, the complete pipeline was proposed that is able to automatically classify human oocytes into three classes MI, MII, and PI based on the oocytes’ microscopic image.

The Belt and Road Initiative in Nepal: Potential impacts and implications

The Belt and Road Initiative is emerging as a global phenomenon with a potential of bearing on almost all nations on Earth in different ways. In this context, this article, generally, is an attempt to demonstrate that impacts and implications of the BRI in Nepal will be governed by important national (Chinese) and international factors. Specifically, it argues that factors that have high developmental potential for Nepal include China's persistent rise and concomitant creation of the multipolar world, China's upholding of its international polices and the BRI principles, reproduction of the BRI experiences of other countries in Nepal, China's continuous pursuit of its socialism-aspiring path of development, its potential embarkment on a predominantly socialist path in the future. It argues that China’s future embarkment on a predominantly capitalist path can have ambivalent impacts ranging from highly appropriative to highly supportive. Likewise, the US–China conflict can also have ambivalent impacts ranging from boosting of Nepal’s negotiating power between the two powers to compromising of Nepal’s sovereignty and stability. The US–China conflict can also push China to treat Nepal as strategically highly important and, therefore, as a favored nation, pouring massive investments. It shows that the Chinese investment will enhance Nepal’s status vis-a-vis the US, the EU and India by cutting down its age-long dependencies on them; and will be conducive to the geographically-distributed development and capital accumulation.

P-117 The reason why direct cleavage and rapid cleavage should be differentiated

Abstract Study question Direct cleavage (DC) and Rapid cleavage (RaC) both appear to be 1 cell divided into 3 (or more) cells, should they be distinguished? Summary answer Since blastomeres by RaC had higher developmental potential than blastomeres by DC, and since many RaC embryos had “normal” blastomere, the two should be distinguished. What is known already DC, in which one cell divides into three (or more) cells without the two-cell stage, and RaC, in which one or two cells divide rapidly after the short two-cell stage (The former lead to segregation of chromosomes into three cells, while the latter leads to inadequate DNA replication), are both often recorded as “DC”, but when classified in detail, there are reports that the blastocyst development rate is higher in RaC embryos. However, the reason for this has not been clarified. Study design, size, duration This was a retrospective study of 643 embryos collected and cultured for at least 5 days in 2020 at our clinic. The embryos were time-lapse monitored by EmbryoScope (Vitrolife, Sweden) and classified into three groups according to the style of first division. Participants/materials, setting, methods The embryos were classified as follows: DC group: embryos that have divided into three (or more) cells without a two-cell stage; RaC group: embryos that rapidly (<5 hours) divided one (or both) cells after the 2-cell stage; Normal cleavage (NC) group: embryos that had a 2-cell stage for more than 5 hours. The subsequent development of blastomeres of all embryos was observed and whether they participated in blastocysts or not was recorded. Main results and the role of chance Of the embryos, 63 were in the DC group and 199 were in the RaC group (173 of which had one rapidly dividing blastomere and 26 of which had two). The blastocyst participation rate of blastomeres was 4.4% in the DC group, 19.5% of rapidly dividing blastomeres and 61.6% of normal blastomeres in the RaC group, with significant differences between all groups (p < 0.01). The good blastocyst (4BC or higher in Gardner grade) development rate was 4.8% (3/63) in the DC group, 40.5% (70/173) in the RaC group with one rapidly dividing blastomere (RaC1 group), 19.2% (5/26) in the RaC group with two such blastomeres (RaC2 group), showing a significant difference between the DC and RaC1 groups (P < 0.01). The live birth rate for single blastocyst transfer was 50% (1/2) in the DC group, 27.8% (5/18) in the RaC1 group, and 100% (1/1) in the RaC2 group. (Note that there were 381 in the NC group, and the above rates were 76.5%, 63.3% (241/381), and 29.3% (22/75), respectively) Limitations, reasons for caution This study examined only the style of first division and did not consider second and subsequent divisions. In addition, as of 2020, the clinical use of PGT-A is not approved in principle in Japan, and the subject embryos have not undergone chromosome analysis. Wider implications of the findings The reason for the higher blastocyst development rate in RaC embryos was indicated by the difference in blastocyst participation rates of the respective blastomeres and the presence of normal blastomeres, but since live births were obtained in both embryos, so these irregular divisions would not completely compromise the blastocyst euploidy. Trial registration number not applicable

O-039 Fertilization: Mechanisms of pronuclear migration and genome unification

Abstract Most early human embryos contain aneuploid cells. Aneuploidy often arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. I will present data showing how the parental genomes cluster with nucleoli in each pronucleus in human and bovine zygotes. Parental genome clustering is required for the reliable unification of the parental genomes after fertilization. During the migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in close proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes upon nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei that are incompatible with healthy embryonic development. In summary, our data reveal why nucleolar clustering correlates with the formation of healthy euploid embryos. In addition, we used automated analysis of nucleolar trajectories and clustering. Our results support the use of nucleolar clustering as a parameter to identify zygotes with higher developmental potential.

An annotated human blastocyst dataset to benchmark deep learning architectures for in vitro fertilization

Medical Assisted Reproduction proved its efficacy to treat the vast majority forms of infertility. One of the key procedures in this treatment is the selection and transfer of the embryo with the highest developmental potential. To assess this potential, clinical embryologists routinely work with static images (morphological assessment) or short video sequences (time-lapse annotation). Recently, Artificial Intelligence models were utilized to support the embryo selection procedure. Even though they have proven their great potential in different in vitro fertilization settings, there is still considerable room for improvement. To support the advancement of algorithms in this research field, we built a dataset consisting of static blastocyst images and additional annotations. As such, Gardner criteria annotations, depicting a morphological blastocyst rating scheme, and collected clinical parameters are provided. The presented dataset is intended to be used to train deep learning models on static morphological images to predict Gardner’s criteria and clinical outcomes such as live birth. A benchmark of human expert’s performance in annotating Gardner criteria is provided.

The variation of total flavonoids, anthocyanins and total phenols in Vaccinium uliginosum fruits in Changbai Mountain of China is closely related to spatial distribution

BACKGROUND: The fruit of Vaccinium uliginosum is a natural berry resource that is rich in polyphenols, flavonol glycosides, anthocyanins, and other active substances, indicating its high developmental potential. However, research on V. uliginosum is limited, with no literature available to clarify the germplasm resources suitable for breeding. OBJECTIVE: This study aimed to evaluate the contents of total flavonoids (TF), total anthocyanins (TA), and total phenols (TP) in 10 different populations of V. uliginosum from the Changbai Mountains, China, and investigate the correlation between these functional components and spatial distribution. METHODS: The components and contents of TA, TF, and TP were determined using mass spectrometry, high-performance liquid chromatography, and the Folin–Ciocalteu method, respectively. RESULTS: A total of 15 anthocyanins were detected, and the content of Mal-glu, Pet-glu, and Del-glu was the highest among these anthocyanins. The TF and TA and TP contents were highest in the DFHI and LJII populations, respectively, which can be reasonably developed as excellent populations. The TF content of sample DFHI-8, TA content of LJIII-1 and TP content of LJIII-4 were higher than other samples, which can be used as important breeding germplasm. The content of TF is positively correlated with altitude, while the content of TA and TP is bidirectional, which is positively correlated at 740–838 m and negatively correlated at >838 m. CONCLUSIONS: Significant differences in the contents of TF, TA, and TP in V. uliginosum fruit were found among and within populations, and there was a certain correlation between these contents and their spatial distribution.

Prediction of live birth - selection of embryos using morphokinetic parameters.

The goal of assisted reproduction is for a couple treated with IVF techniques to end the treatment by giving birth to a healthy baby. A neccessary presumption for success is the identification of the best embryo with high implantation and developmental potential. One option is to select an euploid embryo by invasive preimplantaion genetic testing for aneuploidy (PGT-A) or it is possible to select the best embryo by non-invasive time-lapse monitoring (TLM), specifically based on morphokinetic parameters and morphological markers that are able to identify an embryo with high developmental potential. The study involved a total of 1060 embryos (585 euploid and 475 aneuploid embryos after PGT-A) with good morphology from 329 patients in the period 01/2016-10/2021. All embryos were cultured in a time-lapse incubator, trophectoderm (TE) cells biopsies for PGT-A examination were performed on day 5 (D5) or day 6 (D6) of culture. During the study period, 225 frozen embryo transfers (FET) of one euploid embryo were performed. Based on the treatment outcome, the embryos were divided into 2 groups - euploid embryos, which led to the birth of a healthy child, and euploid embryos that did not show fetal heartbeat (FHB) after FET. Based on the statistical analysis of the embryos without implantation and the embryos with live birth, it is clear that the morphokinetic parameters t5 (time of division into 5 cells) and tSB (time of start of blastulation) are significantly different. The results suggest that of the morphokinetic parameters tSB and t5 are predictive indicators for selecting an embryo with high developmental potential and with a high probability of achieving the birth of a healthy child.

Cleavage kinetics is a better indicator of embryonic developmental competency than brilliant cresyl blue staining of oocytes

In vitro production of embryos (IVP) is a valuable technology to produce embryos of high genetic value. Despite advances in IVP, the efficiency of culture systems remains low. One method to increase IVP success is the early selection of oocytes or embryos that may have greater developmental potential. Here, we investigated two methods of selection, namely BCB staining and cleavage kinetics, both individually and in conjunction, for improved developmental outcomes in vitro. We hypothesized that a synergistic use of both BCB staining and cleavage kinetics would result in identification of embryos of greater developmental potential. The selection of oocytes by BCB staining does select for those oocytes with higher developmental potential, as noted by a greater blastocyst development between BCB positive (32.6%) and BCB negative (22.0%) on day 8 post-fertilization. However, the utilization of BCB staining and cleavage kinetics in tandem resulted in a complete masking of the effect observed when using BCB alone. We obtained the highest proportion of blastocyst development per selection group using cleavage kinetics alone, in which 53.1% of embryos grouped as Fast produced a blastocyst, which was significantly different from the three other groups (Fast+, Slow, not cleaved). We observed, however, that the separation of embryos by cleavage kinetics did not predict their survival to cryopreservation. In conclusion, in standard culture systems, cleavage kinetics is an effective method for the selection of embryos with increased developmental potential to develop blastocysts, however, it may not be effective to select healthy embryos for transfer following cryopreservation.

Morphometric assessment of blastocysts: relationship with the ongoing pregnancy rate

To explore a morphometric grading system for blastocysts that is associated with ongoing pregnancy. Cross-sectional study. None. All consecutive vitrified blastocysts at our center from July 2018 to November 2021 that were transferred in single blastocyst transfer cycles until January2022. None. The ongoing pregnancy rate after a single vitrified-warmed blastocyst transfer. Interobserver agreement on morphometric values among embryologists. Three morphometric variables (blastocyst diameter, area of inner cell mass [ICM], and the estimated trophectoderm cell count) were used to evaluate the expansion, ICM, and trophectoderm morphology. During the study period, 585 blastocysts were involved in this study. Of the 3 morphometric variables, ICM area (per 500 μm2, adjusted odds ratio, 1.19; 95% confidence interval, 1.09-1.30) and estimated trophectoderm cell count (per 10 cells, adjusted odds ratio, 1.25; 95% confidence interval, 1.12-1.39) were significantly associated with the ongoing pregnancy rate after adjustment for confounding factors. The ongoing pregnancy rate was 2.0% (1/49) with an ICM area of <2,500 μm2 and the estimated trophectoderm cell count <70. The ongoing pregnancy rate reached 47.8% (22/46) when the ICM area and the estimated trophectoderm cell count were >3,500 μm2 and >110, respectively. Interobserver agreement on the blastocyst diameter, ICM area, and the estimated trophectoderm cell count was excellent-to-good among 5 embryologists (intraclass correlation coefficients: 0.99, 0.87, and 0.91, respectively). Morphometric values of ICM and trophectoderm are promising predictors of pregnancy success. The high reproducibility suggests that the morphometric variables will contribute to identifying blastocysts with the highest developmental potential as well as those that will not result in a successful pregnancy.

P-528 Time-lapse monitoring: an adjunct tool to select embryos for preimplantation genetic testing

Abstract Study question Does embryo aneuploidy have any impact on embryo morphokinetic events in a time-lapse imaging (TLI) culture system? Summary answer Aneuploidy had a significant impact on early and late embryo morphokinetic events. Mosaic and aneuploid embryos behaved similarly in terms of morphokinetics. What is known already The preimplantation genetic testing (PGT) emerged as an approach to analyse embryos’ DNA for determining genetic abnormalities, distinguishing chromosomally normal embryos, considered to have high developmental potential, from their aneuploid siblings. Although shown to be efficient and clinically relevant, the invasive nature of the technique limits its success. Given the challenges associated with invasive embryo biopsy, ongoing studies are now seeking for non-invasive assessments of the embryo DNA. Timelapse imaging (TLI) may allow the identification of morphokinetics events affected by aneuploidy, which could be a powerful tool for improving embryo selection for transfer, without the detrimental effects of embryo biopsy. Study design, size, duration This case-control study was performed in a private university–affiliated IVF center, between 2019 March and 2020 December. Kinetic data were analyzed in 957 embryos, derived from 316 patients undergoing ICSI cycle with PGT-A. Kinetic markers from the point of insemination were recorded. Generalized linear models followed by Bonferroni post hoc were used to compare timing of specific events in euploid (n = 352), aneuploid (n = 583) and mosaic embryos (n = 22). The post hoc achieved power was &amp;gt; 80%. Participants/materials, setting, methods Embryos were cultured in the EmbryoScope incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), morulae (tM), start of blastulation (tSB) and blastulation (tB). Durations of second and third cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1, s2, and s3 were calculated. The KIDScore ranking was recorded. Main results and the role of chance Aneuploid embryos showed significantly longer tPNf, t2, t3, t4, t6, t7, t8, tM, tSB, tB, s2 and s3 compared to euploid embryos. The KIDScore day 5 also rank was significantly lower in aneuploid embryos compared to euploid ones. As for mosaic embryos no significant differences were observed in the comparisons of means of morphokinetic events and KIDScore rank, but regression analyses showed an increased chance of mosaicism among embryos with longer tPNf, t2, t3, t6, t7, tM, tB, as well as a inverse association with KIDScore rank, when compared with euploid embryos. Embryos were hierarchically distributed into quartiles according to the KIDScore rank: Q1 ≤3.9, Q2 4-5.6, Q3 5.7-7.5, and Q4 ≥7.6. Euploidy incidence was significantly different among the KIDScore quartiles, in which the percentage of euploidy increased directly proportional to the kidscore rank. For patients &amp;gt;35 years old, the logistic regression analysis demonstrated that comparing with embryos in the Q4, embryos in the Q1, Q2, and Q3 have a decreased chance of being euploid. For patients ≤35 years old, comparing with embryos in the Q4, embryos with KIDScore D5 in the Q1 and Q2, but not in Q3, have a decreased chance of being euploid. Limitations, reasons for caution Our study is limited by its retrospective nature and small casuistic, despite adequate power has been achieved. The reason for the lack of consistency with some previous findings can be explained by variations in culture systems, studied population, embryo stage for biopsy and wide range of tests used for PGT. Wider implications of the findings Our evidence suggests that TLI monitoring may be an adjunct approach to select embryos for PGT, however, cautious investigation is still needed. The mechanism by which embryo aneuploidy may alter the cleavage timing of embryos is undefined, but disruptions of mitosis, cell-cell interactions and blastocyst differentiation are possible candidates. Trial registration number not applicable

Improved pregnancy outcomes from mosaic embryos with lower mtDNA content: a single-center retrospective study

Research questionThe purpose of this study is to investigate whether the mitochondrial DNA (mtDNA) content can reflect the state of mosaic embryos. DesignThe study included 1669 blastocysts derived from 394 PGT-A cycles between January 2018 and December 2020, in which preimplantation genetic testing for aneuploidy was performed and mtDNA content was determined. The standard deviation (SD) of whole genomic sequencing data was calculated for quality control. mtDNA content was measured as the proportion of mtDNA to genomic DNA. 1558 blastocysts with SD values less than 4.0 and mtDNA values less than 0.4% were selected for statistical analysis. ResultsThe mtDNA content of the PGT mosaic group was significantly higher than that of the PGT normal group (P < 0.001). Twenty-six mosaic embryos were transferred, and the results were as follows: 2 out of 26 had undergone a spontaneous miscarriage, 15 were not pregnant, and 9 resulted in a live birth. There were significant differences in the mtDNA content between the miscarriage/non-pregnancy group and the live birth group (**P < 0.01; ***P < 0.001). There was no mosaic embryo with more than 0.157% mtDNA content found in the live birth group. ConclusionsThis study demonstrates that mtDNA analysis has the ability to identify mosaic embryos with high developmental potential. It can be a valuable supplementary index for the selection of mosaic embryos for transfer. Larger studies with a greater sample size will further our understanding of the relationships between metabolic activity and mosaicism.

Abstract 401: Single-cell transcriptome and chromatin sequencing uncover gene expression and gene regulatory patterns associated with enzalutamide resistance

Abstract Resistance to androgen receptor-targeted therapy due to tumor heterogeneity and clonal evolution is a key challenge for improving prostate cancer outcomes. Despite this, the transcriptomic and chromatin accessibility changes contributing to the emergence of resistance remain incompletely understood at the level of individual cells. Using single-cell assays for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide, we previously identified pre-existing and persistent cell subpopulations that possess regenerative potential when subjected to treatment. Here we analyze the chromatin and transcriptomes of these single cells to characterize their gene regulation and gene expression trajectories. We present evidence of a model of enzalutamide resistance emergence in which the pre-existing and treatment-persistent cells regenerate the bulk of resistant cells. This process is underpinned by chromatin reprogramming that increases the overall relaxation of chromatin upon resistance. We show that the reprogramming of the chromatin further differentially contributes to transcription factor-mediated transcriptional reprogramming via DNA motif exposure in different cell subpopulations. For example, in the treatment-persistent cells, we identify chromatin configurations characterized by the exposure of DNA motifs for GATA2, RELA (a NFkB subunit), CREB1, and E2F1. Pre-existing and treatment-persistent cells consistently display transcriptional features of high developmental potential and RNA velocity analysis identifies them as precursors of cell populations that arise from enzalutamide treatment. We also analyze the pre-existing and treatment-persistent cells in spatial transcriptomics of prostate cancer patient specimens based on their characteristic gene expression profiles. We find these cells to be enriched in cancerous regions of the tissue but also detect them within apparent benign regions, which has potential implications for treatment choice. In summary, we show patterns of gene expression regulation in preclinical models and patient samples that uncover mechanisms of resistance to androgen receptor-targeted therapy in prostate cancer. Citation Format: Sinja Taavitsainen, Nikolai Engedal, Shaolong Cao, Florian Handle, Andrew Erickson, Stefan Prekovic, Daniel Wetterskog, Teemu Tolonen, Elisa M. Vuorinen, Antti Kiviaho, Reetta Nätkin, Tomi Häkkinen, Wout Devlies, Sallamari Henttinen, Roosa Kaarijärvi, Mari Lahnalampi, Heidi Kaljunen, Karolina Nowakowska, Heimo Syvälä, Merja Bläuer, Paolo Cremaschi, Frank Claessens, Tapio Visakorpi, Teuvo L. Tammela, Teemu Murtola, Kirsi J. Granberg, Alastair D. Lamb, Kirsi Ketola, Ian G. Mills, Gerhardt Attard, Wenyi Wang, Matti Nykter, Alfonso Urbanucci. Single-cell transcriptome and chromatin sequencing uncover gene expression and gene regulatory patterns associated with enzalutamide resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 401.

Genome architecture and totipotency: An intertwined relation during early embryonic development.

Chromosomes are not randomly packed and positioned into the nucleus but folded in higher-order chromatin structures with defined functions. However, the genome of a fertilized embryo undergoes a dramatic epigenetic reprogramming characterized by extensive chromatin relaxation and the lack of a defined three-dimensional structure. This reprogramming is followed by a slow genome refolding that gradually strengthens the chromatin architecture during preimplantation development. Interestingly, genome refolding during early development coincides with a progressive loss of developmental potential suggesting a link between chromatin organization and cell plasticity. In agreement, loss of chromatin architecture upon depletion of the insulator transcription factor CTCF in embryonic stem cells led to the upregulation of the transcriptional program found in totipotent cells of the embryo, those with the highest developmental potential. This essay will discuss the impact of genome folding in controlling the expression of transcriptional programs involved in early development and their plastic-associated features.